The novel FAT4 activator jujuboside A suppresses NSCLC tumorigenesis by activating HIPPO signaling and inhibiting YAP nuclear translocation.

FAT atypical cadherin 4 (FAT4) has been identified as a tumor suppressor in lung cancers. However, no agent for lung cancer treatment targeting FAT4 has been used in the clinic. Jujuboside A (JUA) is a major active compound in Semen Ziziphi Spinosae. Semen Ziziphi Spinosae is a traditional Chinese herbal medicine used clinically for tumor treatment to improve patients' quality of life. However, the anti-lung cancer activity and the underlying mechanisms of JUA are not yet fully understood. Here, we demonstrated the anti-lung cancer activity of JUA in two lung cancer mice models and three non-small cell lung cancer (NSCLC) cell lines, and further illustrated its underlying mechanisms. JUA suppressed the occurrence and development of lung cancer and extended mice survival in vivo, and suppressed NSCLC cell activities through cell cycle arrest, proliferation suppression, stemness inhibition and senescence promotion. Moreover, JUA directly bound with and activated FAT4, subsequently activating FAT4-HIPPO signaling and inhibiting YAP nuclear translocation. Knockdown of FAT4 diminished JUA's effects on HIPPO signaling, YAP nuclear translocation, cell proliferation and cellular senescence. In conclusion, JUA significantly suppressed NSCLC tumorigenesis by regulating FAT4-HIPPO-YAP signaling. Our findings suggest that JUA is a novel FAT4 activator that can be developed as a promising NSCLC therapeutic agent targeting the FAT4-HIPPO-YAP pathway.

The flavonoid quercetin reduces cell migration and increases NIS and E-cadherin mRNA in the human thyroid cancer cell line BCPAP.

Thyroid cancer is the most frequent cancer of the endocrine system. Most patients are treated with thyroidectomy followed by radioiodine therapy. However, in part of the patients, a reduction of the sodium-iodide symporter (NIS) occurs, rendering radioiodine therapy ineffective. Moreover, epithelial-mesenchymal transition (EMT) may occur, leading to more aggressive and invasive features. Herein, we evaluated the effect of the flavonoid quercetin on EMT and NIS expression in BCPAP, a papillary thyroid carcinoma cell line. BCPAP was treated with 100 µM quercetin for 24 h and cell viability, apoptosis, EMT markers and NIS were evaluated. Quercetin decreased cell viability by enhancing apoptosis. The flavonoid also reduced matrix metalloproteinase 9 and increased E-cadherin mRNA levels, inhibiting BCPAP adhesion and migration. Additionally, quercetin increased NIS expression and function. Thus, our results suggest that quercetin could be useful as adjuvant in thyroid cancer therapy, inducing apoptosis, reducing invasion and increasing the efficacy of radioiodine therapy.

Enhanced endothelial barrier function by monoclonal antibody activation of vascular endothelial cadherin.

Excessive vascular permeability occurs in inflammatory disease processes. Vascular endothelial cadherin (VE-cadherin) is an adhesion protein that controls vascular permeability. We identified monoclonal antibodies (mAbs) to human VE-cadherin that activate cell adhesion and inhibit the increased permeability of endothelial cell monolayers induced by thrombin receptor activator peptide-6 (TRAP-6). Two mAbs, 8A12c and 3A5a, reduce permeability, whereas an inhibitory mAb, 2E11d, enhances permeability. Activating mAbs also reduce permeability induced by tumor necrosis factor-α (TNF-α) and vascular endothelial cell growth factor (VEGF). The activating mAbs also stabilize the organization of the adherens junctions that are disrupted by TRAP-6, VEGF, or TNF-α. The activating mAbs act directly on the adhesive function of VE-cadherin because they did not block the accumulation of actin filaments stimulated by TRAP-6 and enhance physical cell-cell adhesion of VE-cadherin-expressing tissue culture cells. Therefore, VE-cadherin function can be regulated at the cell surface to control endothelial permeability.NEW & NOTEWORTHY Excessive vascular permeability is a serious complication of many inflammatory disease conditions. We have developed monoclonal antibodies that inhibit increases in endothelial monolayer permeability induced by several signaling factors by activating VE-cadherin mediated adhesion and stabilizing cell junctions. These antibodies and/or the mechanisms they reveal may lead to important therapeutics to treat vascular leakiness and inflammation.

The peptide mimicking small extracellular ring domain of CD82 inhibits epithelial-mesenchymal transition by downregulating Wnt pathway and upregulating hippo pathway.

We have previously demonstrated that the peptide mimicking small extracellular ring domain of CD82 (CD82EC1-mP) could inhibit tumor cell motility and metastasis. However, its acting mechanism is not understood. Here, we reported that the cell motility-inhibitory function of CD82EC1-mP was involved in the downregulation of epithelial-mesenchymal transition (EMT). Both vimentin and E-cadherin are EMT makers. We found that CD82EC1-mP could inhibit the expression of vimentin, but promot the expression of E-cadherin, suggesting that CD82EC1-mP suppressed EMT. Hippo/YAP and Wnt/ß-catenin are both key signal pathways that regulate the EMT process. The futher studies showed that CD82EC1-mP couled activate GSK3ß, promote the phosphorylation of ß-catenin, and inhibit the ß-catenin nuclear location. Moreover, CD82EC1-mP couled activate Hipoo kinase cascade, promote the phosphorylation of YAP, and inhibit the YAP nuclear location. These results suggested that CD82EC1-mP inhibited invation and matestasis via inhibiting EMT through downregulating Wnt pathway and upregulating Hippo pathway.

3-deazaneplanocin A protects against cisplatin-induced renal tubular cell apoptosis and acute kidney injury by restoration of E-cadherin expression.

3-deazaneplanocin A (3-DZNeP) has been used as an inhibitor of enhancer of zeste homolog 2 (EZH2). Here, we explore the role and underlying mechanisms action of 3-DZNeP in abrogating cisplatin nephrotoxicity. Exposure of cultured mouse renal proximal tubular epithelial cells (mTECs) to cisplatin resulted in dose and time-dependent cleavage of caspase-3, decrease of cell viability, and increase of histone H3 lysine 27 trimethylation (H3K27me3), whereas expression levels of EZH2, a major methyltransferase of H3K27me3, were not affected. Treatment with 3-DZNeP significantly inhibited cisplatin-induced activation of caspase-3, apoptosis, loss of cell viability but did not alter levels of EZH2 and H3K27me3 in cultured mTECs. 3-DZNeP treatment did not affect activation of extracellular signal-regulated kinase (ERK) 1/2, p38 or c-Jun N-terminal kinases (JNK) 1/2, which contribute to renal epithelial cell death, but caused dose-dependent restoration of E-cadherin in mTECs exposed to cisplatin. Silencing of E-cadherin expression by siRNA abolished the cytoprotective effects of 3-DZNeP. In contrast, 3-DZNeP treatment potentiated the cytotoxic effect of cisplatin in H1299, a non-small cell lung cancer cell line that expresses lower E-cadherin levels. Finally, administration of 3-DZNeP attenuated renal dysfunction, morphological damage, and renal tubular cell death, which was accompanied by E-cadherin preservation, in a mouse model of cisplatin nephrotoxicity. Overall, these data indicate that 3-DZNeP suppresses cisplatin-induced tubular epithelial cell apoptosis and acute kidney injury via an E-cadherin-dependent mechanism, and suggest that combined application of 3-DZNeP with cisplatin would be a novel chemotherapeutic strategy that enhances the anti-tumor effect of cisplatin and reduces its nephrotoxicity.

Concurrent exercise improves insulin resistance and nonalcoholic fatty liver disease by upregulating PPAR-γ and genes involved in the beta-oxidation of fatty acids in ApoE-KO mice fed a high-fat diet.

OBJECTIVE: To emphasize the mechanism of concurrent exercise effect on lipid disorders in insulin resistance (IR) and nonalcoholic fatty liver disease (NAFLD). MATERIALS AND METHODS: Twenty male ApoE knockout mice were randomly divided into two groups: HFD group (n = 10) fed a high fat diet, and HFDE group (n = 10) with high-fat diet intervention for 12 weeks and swimming exercise. Other ten healthy male C57BL/6 J mice were fed a normal diet, and included as control group. Retro-orbital blood samples were collected for biochemical analysis. Oil red O staining of liver tissues was performed to confirm the exercise effect. Western blotting was performed to evaluate the expressions of PPAR-γ, CPT-1, MCAD. RESULTS: The levels of TG, TC, LDL, FFA, FIN, FPG and Homa-IRI in the HFD group were significantly higher than ND group, while these were markedly decreased in the HFDE group compared with HFD group. The Oil Red O staining of liver samples further confirmed the exercise effect on the change of lipid deposition in the liver. Western blotting showed increased expressions of PPAR-γ, CPT-1, MCAD induced by high fat diet were significantly downregulated by exercise. CONCLUSION: A concurrent 12-week exercise protocol alleviated the lipid metabolism disorders of IR and NAFLD, probably via PPAR-γ/CPT-1/MCAD signaling.

C1q tumor necrosis factor-related protein 9 in atherosclerosis: Mechanistic insights and therapeutic potential.

C1q tumor necrosis factor-related protein 9 (CTRP9), a newly discovered adipokine, is the closest paralog of adiponectin. After proteolytic cleavage, it can release the globular domain (gCTRP9) that serves as the major circulatory isoform. Upon binding to adiponectin receptor 1 (AdipoR1) and N-cadherin, CTRP9 can activate a variety of signaling pathways to regulate glucose and lipid metabolism, vascular relaxation and cell differentiation. Circulating CTRP9 levels are significantly decreased in patients with coronary atherosclerosis disease. Data obtained from in vitro experiments and animal models suggest that CTRP9 exerts an atheroprotective effect by altering multiple pathological processes involved in atherosclerosis, including inflammation, foam cell formation, endothelial dysfunction, insulin resistance, and vascular smooth muscle cell dedifferentiation, proliferation and migration. In this review, we summarize the latest advances regarding the roles of CTRP9 in atherosclerosis with an emphasis on its potential as a novel therapeutic target in cardiovascular disease.

Dihydroartemisinin up-regulates VE-cadherin expression in human renal glomerular endothelial cells.

The antimalarial agent dihydroartemisinin (DHA) has been shown to be anti-inflammatory. In this study, we found that DHA increased the expression of the junctional protein vascular endothelial (VE)-cadherin in human renal glomerular endothelial cells. In addition, DHA inhibited TGF-ß RI-Smad2/3 signalling and its downstream effectors SNAIL and SLUG, which repress VE-cadherin gene transcription. Correspondingly, DHA decreased the binding of SNAIL and SLUG to the VE-cadherin promoter. Together, our results suggest an effect of DHA in regulating glomerular permeability by elevation of VE-cadherin expression.

Hedyotis diffusa Willd suppresses metastasis in 5­fluorouracil­resistant colorectal cancer cells by regulating the TGF­ß signaling pathway.

Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive tract, and threatens the survival and health of patients with CRC. Chemotherapy remains one of the main therapeutic approaches for patients with CRC; however, drug resistance limits the long­term use. CRC cells with multi­drug resistance (MDR) exhibit increased survival times and metastatic potential, which may lead to the recurrence and metastasis of CRC. In addition, MDR is one of the major causes of chemotherapy failure in clinical treatment. Hedyotis diffusa Willd (HDW) has been used in the treatment of inflammation­associated diseases and malignant tumors, including CRC. The authors previously demonstrated that HDW could reverse MDR in CRC cells; however, its underlying mechanism, particularly in MDR­associated metastasis, remains to be elucidated. In the present study, the drug­resistant CRC cell line HCT­8/5­fluorouracil (5­FU) was used to investigate the effect of HDW on the growth and metastasis of cancer cells. Cell viability was assessed using the MTT assay. Cell adhesion potential was evaluated using adhesion experiments. Cell migration was assessed using wound healing and Transwell assays. The mRNA and protein expression levels of crucial factors in the transforming growth factor­ß (TGF­ß) signaling pathway, including TGF­ß, Mothers against decapentaplegic homolog 4 (SMAD4), neural (N)­cadherin, and epithelial (E)­cadherin, were analyzed using the reverse transcription­semi­quantitative polymerase chain reaction and western blotting, respectively. The results demonstrated that the HCT­8/5­FU cell line was more resistant to 5­FU and thus can be used as the resistant cell model. HDW was able to inhibit the viability, and adhesive, migratory and invasion potential of the HCT­8/5­FU cells. In addition, HDW was able to downregulate the expression of TGF­ß, SMAD4 and N­cadherin, and upregulate E­cadherin, at the gene and protein level. In conclusion, the results demonstrated that HDW may suppress the metastasis of 5­FU­resistant CRC cells via regulation of the TGF­ß signaling pathway, which was also considered to be one of the underlying mechanisms of its anti­CRC effect.

The ROCK inhibitor, thiazovivin, inhibits human corneal endothelial­to­mesenchymal transition/epithelial­to­mesenchymal transition and increases ionic transporter expression.

Corneal diseases exhibit a high prevalence and are prone to cause blindness; furthermore, maintaining the morphology and ionic transporter expression in corneal endothelial cells (CECs) is crucial for treatment of these diseases. This study aimed to investigate the effects of the novel Rho associated coiled-coil containing protein kinase (ROCK) inhibitor, thiazovivin (2,4­disubstituted thiazole, TZV), on human corneal endothelial­to­mesenchymal transition/epithelial­to­mesenchymal transition (EndMT/EMT), cell morphology, junction proteins and ionic transporter expression in human CECs (HCECs) in vitro and then to clarify the mechanisms of action of TZV. In the present study, primary HCECs were cultured in vitro and passaged. The expression levels of adhesion proteins (E­cadherin and N­cadherin), the EndMT/EMT marker, α smooth muscle  actin (α­SMA), the tight junction protein, Zonula occludens-1 (ZO­1), and the ionic transporter, Na+/K+­ATPase, were detected by immunofluorescence. The proliferative ability of the HCECs was determined by CCK-8 assay. The mRNA expression of the EndMT/EMT­inducing gene, Snail, was examined by RT­PCR. The protein expression levels of ROCK1/2 were evaluated by western blot analysis. The HCECs were cultured with TZV at various concentrations (2, 4, or 6 µM) for different periods of time (24 or 48 h). We found that the the cell states of the HCECs co­cultured with 4 µM TZV for 48 h reached the optimum, and corneal EndMT/EMT was inhibited, as evidenced by the significantly upregulated expression of ZO­1 and E­cadherin, and the markedly downregulated expression of N­cadherin and α­SMA. Furthermore, the cells exhibited a normal, tightly connected hexagonal or pentagonal morphology. Additionally, the protein expression of ROCK1/2 and the mRNA expression of Snail were significantly decreased. However, there was no significant difference between the TZV­treated and the control groups as regards HCEC proliferative ability. These findings suggested that the ROCK inhibitor, TZV (4 µM), was effective in improving the morphology, cell junctions and ionic transporter expression of HCECs by inhibiting EndMT/EMT, but had no effect on HCEC proliferation.

Effect of silencing the T­Box transcription factor TBX2 in prostate cancer PC3 and LNCaP cells.

T­Box (TBX)­2 is a member of the T­box gene family, which is aberrantly expressed in numerous types of malignant tumors, and has previously been demonstrated to be conducive to tumor progression by acting as a transcription factor. However, specific information regarding the expression and function of TBX2 in prostate cancer cells remains to be elucidated. The present study demonstrated that silencing of TBX2 by TBX2 small interfering RNA inhibited cell proliferation and promoted cell senescence. It was demonstrated that knockdown of TBX2 inhibited cell metastatic abilities by upregulating E­cadherin and downregulating N­cadherin, Vimentin and fibronectin. In addition, the expression of TBX2 in prostate cancer tissues and tumor adjacent tissues was detected by immunohistochemistry. The results indicated that the expression rates of TBX2 were significantly increased in the cancerous tissues, compared with the healthy tumor adjacent tissue, and TBX2 increased staining was associated with the clinical stage and pathological grade. The findings of the present study therefore suggest that TBX2 expression is markedly increased in prostate cancer and TBX2 may act as a potential beneficial therapeutic target for the future treatment of prostate cancer.

Dual histone reader ZMYND8 inhibits cancer cell invasion by positively regulating epithelial genes.

Enhanced migratory potential and invasiveness of cancer cells contribute crucially to cancer progression. These phenotypes are achieved by precise alteration of invasion-associated genes through local epigenetic modifications which are recognized by a class of proteins termed a chromatin reader. ZMYND8 [zinc finger MYND (myeloid, Nervy and DEAF-1)-type containing 8], a key component of the transcription regulatory network, has recently been shown to be a novel reader of H3.1K36Me2/H4K16Ac marks. Through differential gene expression analysis upon silencing this chromatin reader, we identified a subset of genes involved in cell proliferation and invasion/migration regulated by ZMYND8. Detailed analysis uncovered its antiproliferative activity through BrdU incorporation, alteration in the expression of proliferation markers, and cell cycle regulating genes and cell viability assays. In addition, performing wound healing and invasion/migration assays, its anti-invasive nature is evident. Interestingly, epithelial-mesenchymal transition (EMT), a key mechanism of cellular invasion, is regulated by ZMYND8 where we identified its selective enrichment on promoters of CLDN1/CDH1 genes, rich in H3K36Me2/H4K16Ac marks, leading to their up-regulation. Thus, the presence of ZMYND8 could be implicated in maintaining the epithelial phenotype of cells. Furthermore, syngeneic mice, injected with ZMYND8-overexpressed invasive breast cancer cells, showed reduction in tumor volume and weight. In concert with this, we observed a significant down-regulation of ZMYND8 in invasive ductal and lobular breast cancer tissues compared with normal tissue. Taken together, our study elucidates a novel function of ZMYND8 in regulating EMT and invasion of cancer cells, possibly through its chromatin reader function.

1α,25-Dihydroxyvitamin D Inhibits the Metastatic Capability of MCF10CA1a and MDA-MB-231 Cells in an In Vitro Model of Breast to Bone Metastasis.

Breast cancer metastasis to the bone continues to be a major health problem, with approximately 80% of advanced breast cancer patients expected to develop bone metastasis. Although the problem of bone metastasis persists, current treatment options for metastatic cancer patients are limited. In this study, we investigated the preventive role of the active vitamin D metabolite, 1α,25-dihydroxyvitamin D (1,25(OH)2D), against the metastatic potential of breast cancer cells using a novel three-dimensional model (rMET) recapitulating multiple steps of the bone metastatic process. Treatment of MCF10CA1a and MDA-MB-231 cells inhibited metastasis in the rMET model by 70% (±5.7%) and 21% (±6%), respectively. In addition, 1,25(OH)2D treatment decreased invasiveness (20 ± 11% of vehicle) and decreased the capability of MCF10CA1a cells to survive in the reconstructed bone environment after successful invasion through the basement membrane (69 ± 5% of vehicle). An essential step in metastasis is epithelial-mesenchymal transition (EMT). Treatment of MCF10CA1a cells with 1,25(OH)2D increased gene (2.04 ± 0.28-fold increase) and protein (1.87 ± 0.20-fold increase) expression of E-cadherin. Additionally, 1,25(OH)2D treatment decreased N-cadherin gene expression (42 ± 8% decrease), a marker for EMT. Collectively, the present study suggests that 1,25(OH)2D inhibits breast cancer cell metastatic capability as well as inhibits EMT, an essential step in the metastatic process.

Inhibition of LSD1 by Pargyline inhibited process of EMT and delayed progression of prostate cancer in vivo.

Recently, lysine-specific demethylase 1 (LSD1) was identified as the first histone demethylase. LSD1 interacted with androgen receptor (AR) and promoted androgen-dependent transcription of target genes, such as PSA, by ligand-induced demethylation of mono- and dimethylated histone H3 at Lys 9 (H3K9). Meanwhile, the phenomenon of epithelial-mesenchymal transition (EMT) had received considerable attention in tumor recurrence and metastasis. This study examined the effect of Pargyline (an inhibitor of LSD1) on the process of EMT in vitro and in vivo. SCID mice were injected subcutaneously with LNCap cells. Pargyline was given intraperitoneally or not after castration (implemented with Bilateral orchidectomy), then PSA levels in serum and tumor were determined to assess time to androgen-independent progression. The results showed that LSD1 expression was up-regulated when PCa progressed to Castration Resistant Prostate Cancer (CRPC). Pargyline reduced LNCap cells migration and invasion ability, and inhibited the process of EMT by up-regulating expression of E-cadherin, and down-regulating expressions of N-cadherin and Vimentin in vitro and in vivo. Although, Pargyline did not change the level of AR, it reduced PSA expression both in vitro and in vivo. Furthermore, Pargyline delayed prostate cancer transition from androgen-dependent to androgen-independent state (CRPC). These findings indicated that inhibition of LSD1 might be a promise adjunctive therapy with androgen deprivation therapy (ADT) for locally advanced or metastatic prostate cancer.

Flavocoxid Protects Against Cadmium-Induced Disruption of the Blood­Testis Barrier and Improves Testicular Damage and Germ Cell Impairment in Mice [corrected].

Cadmium (Cd) causes male infertility. There is the need to identify safe treatments counteracting this toxicity. Flavocoxid is a flavonoid that induces a balanced inhibition of cyclooxygenase (COX)-1 and COX-2 peroxidase moieties and of 5-lipoxygenase (LOX) and has efficacy in the male genitourinary system. We investigated flavocoxid effects on Cd-induced testicular toxicity in mice. Swiss mice were divided into 4 groups: 2 control groups received 0.9% NaCl (vehicle; 1 ml/kg/day) or flavocoxid (20 mg/kg/day ip); 2 groups were challenged with cadmium chloride (CdCl2; 2 mg/kg/day ip) and administered with vehicle or flavocoxid. The treatment lasted for 1 or 2 weeks. The testes were processed for biochemical and morphological studies. CdCl2 increased phosphorylated extracellular signal-regulated kinase (p-ERK) 1/2, tumor necrosis factor (TNF)-α, COX-2, 5-LOX, malondialdehyde (MDA), B-cell-lymphoma (Bcl)-2-associated X protein (Bax), follicle-stimulating hormone (FSH), luteinizing hormone (LH), transforming growth factor (TGF) -ß3, decreased Bcl-2, testosterone, inhibin-B, occludin, N-Cadherin, induced structural damages in the testis and disrupted the blood-testis barrier. Many TUNEL-positive germ cells and changes in claudin-11, occludin, and N-cadherin localization were present. Flavocoxid administration reduced, in a time-dependent way, p-ERK 1/2, TNF-α, COX-2, 5-LOX, MDA, Bax, FSH, LH, TGF-ß3, augmented Bcl-2, testosterone, inhibin B, occludin, N-Cadherin, and improved the structural organization of the testis and the blood-testis barrier. Few TUNEL-positive germ cells were present and a morphological retrieval of the intercellular junctions was observed. In conclusion, flavocoxid has a protective anti-inflammatory, antioxidant, and antiapoptotic function against Cd-induced toxicity in mice testis. We suggest that flavocoxid may play a relevant positive role against environmental levels of Cd, otherwise deleterious to gametogenesis and tubular integrity.

RANKL promotes migration and invasion of hepatocellular carcinoma cells via NF-κB-mediated epithelial-mesenchymal transition.

BACKGROUND: Metastasis accounts for the most deaths in patients with hepatocellular carcinoma (HCC). Receptor activator of nuclear factor kappa B ligand (RANKL) is associated with cancer metastasis, while its role in HCC remains largely unknown. METHODS: Immunohistochemistry was performed to determine the expression of RANK in HCC tissue (n = 398). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to examine the expression of RANK, E-cadherin, N-cadherin, vimentin, Snail, Slug, Twist and MMPs in HCC cells. Wound healing and Transwell assays were used to evaluate cell migration and invasion ability. RESULTS: We found that expression of RANK, the receptor of RANKL, was significantly higher in HCC tumor tissues than in peritumor liver tissues (p<0.001). Constitutive expression of RANK was detected in HCC cell lines, which can be up-regulated when HCC cells were stimulated with RANKL. Notably, in vitro experiments showed that activation of RANKL-RANK axis significantly promoted migration and invasion ability of HCC cells. In addition, RANKL stimulation increased the expression levels of N-cadherin, Snail, and Twist, while decreased the expression of E-cadherin, with concomitant activation of NF-κB signaling pathway. Moreover, administration of the NF-κB inhibitor attenuated RANKL-induced migration, invasion and epithelial-mesenchymal transition of HCC cells. CONCLUSIONS: RANKL could potentiate migration and invasion ability of RANK-positive HCC cells through NF-κB pathway-mediated epithelial-mesenchymal transition, which means that RANKL-RANK axis could be a potential target for HCC therapy.

Angiotensin II induces Fat1 expression/activation and vascular smooth muscle cell migration via Nox1-dependent reactive oxygen species generation.

Fat1 is an atypical cadherin that controls vascular smooth muscle cell (VSMC) proliferation and migration. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (Nox1) is an important source of reactive oxygen species (ROS) in VSMCs. Angiotensin II (Ang II) induces the expression and/or activation of both Fat1 and Nox1 proteins. This study tested the hypothesis that Ang II-induced Fat1 activation and VSMC migration are mediated by Nox1-dependent ROS generation and redox signaling. Studies were performed in cultured VSMCs from Sprague­Dawley rats. Cells were treated with Ang II (1 µmol/L) for short (5 to 30 min) or long term stimulations (3 to 12 h) in the absence or presence of the antioxidant apocynin (10 µmol/L), extracellular-signal-regulated kinases 1/2 (Erk1/2) inhibitor PD98059 (1 µmol/L), or Ang II type 1 receptor (AT1R) valsartan (1 µmol/L). siRNA was used to knockdown Nox1 or Fat1. Cell migration was determined by Boyden chamber assay. Ang II increased Fat1 mRNA and protein levels and promoted Fat1 translocation to the cell membrane, responses that were inhibited by AT1R antagonist and antioxidant treatment. Downregulation of Nox1 inhibited the effects of Ang II on Fat1 protein expression. Nox1 protein induction, ROS generation, and p44/p42 MAPK phosphorylation in response to Ang II were prevented by valsartan and apocynin, and Nox1 siRNA inhibited Ang II-induced ROS generation. Knockdown of Fat1 did not affect Ang II-mediated increases in Nox1 expression or ROS. Inhibition of p44/p42 MAPK phosphorylation by PD98059 abrogated the Ang II-induced increase in Fat1 expression and membrane translocation. Knockdown of Fat1 inhibited Ang II-induced VSMC migration, which was also prevented by valsartan, apocynin, PD98059, and Nox1 siRNA. Our findings indicate that Ang II regulates Fat1 expression and activity and induces Fat1-dependent VSMC migration via activation of AT1R, ERK1/2, and Nox1-derived ROS, suggesting a role for Fat1 downstream of Ang II signaling that leads to vascular remodeling.

A Spodoptera exigua cadherin serves as a putative receptor for Bacillus thuringiensis Cry1Ca toxin and shows differential enhancement of Cry1Ca and Cry1Ac toxicity.

Crystal toxin Cry1Ca from Bacillus thuringiensis has an insecticidal spectrum encompassing lepidopteran insects that are tolerant to current commercially used B. thuringiensis crops (Bt crops) expressing Cry1A toxins and may be useful as a potential bioinsecticide. The mode of action of Cry1A is fairly well understood. However, whether Cry1Ca interacts with the same receptor proteins as Cry1A remains unproven. In the present paper, we first cloned a cadherin-like gene, SeCad1b, from Spodoptera exigua (relatively susceptible to Cry1Ca). SeCad1b was highly expressed in the larval gut but scarcely detected in fat body, Malpighian tubules, and remaining carcass. Second, we bacterially expressed truncated cadherin rSeCad1bp and its interspecific homologue rHaBtRp from Helicoverpa armigera (more sensitive to Cry1Ac) containing the putative toxin-binding regions. Competitive binding assays showed that both Cry1Ca and Cry1Ac could bind to rSeCad1bp and rHaBtRp, and they did not compete with each other. Third, Cry1Ca ingestion killed larvae and decreased the weight of surviving larvae. Dietary introduction of SeCad1b double-stranded RNA (dsRNA) reduced approximately 80% of the target mRNA and partially alleviated the negative effect of Cry1Ca on larval survival and growth. Lastly, rSeCad1bp and rHaBtRp differentially enhanced the negative effects of Cry1Ca and Cry1Ac on the larval mortalities and growth of S. exigua and H. armigera. Thus, we provide the first lines of evidence to suggest that SeCad1b from S. exigua is a functional receptor of Cry1Ca.

Downregulation of OPA3 is responsible for transforming growth factor-ß-induced mitochondrial elongation and F-actin rearrangement in retinal pigment epithelial ARPE-19 cells.

Transforming growth factor-ß signaling is known to be a key signaling pathway in the induction of epithelial-mesenchymal transition. However, the mechanism of TGF-ß signaling in the modulation of EMT remains unclear. In this study, we found that TGF-ß treatment resulted in elongation of mitochondria accompanied by induction of N-cadherin, vimentin, and F-actin in retinal pigment epithelial cells. Moreover, OPA3, which plays a crucial role in mitochondrial dynamics, was downregulated following TGF-ß treatment. Suppression of TGF-ß signaling using Smad2 siRNA prevented loss of OPA3 induced by TGF-ß. Knockdown of OPA3 by siRNA and inducible shRNA significantly increased stress fiber levels, cell length, cell migration and mitochondrial elongation. In contrast, forced expression of OPA3 in ARPE-19 cells inhibited F-actin rearrangement and induced mitochondrial fragmentation. We also showed that Drp1 depletion increased cell length and induced rearrangement of F-actin. Depletion of Mfn1 blocked the increase in cell length during TGF-ß-mediated EMT. These results collectively substantiate the involvement of mitochondrial dynamics in TGF-ß-induced EMT.

Up-regulation of E-cadherin by small activating RNA inhibits cell invasion and migration in 5637 human bladder cancer cells.

Recent studies have reported that chemically synthesized small duplex RNAs complementary to promoters of target genes can specifically induce gene expression in several cancer cell lines. Such dsRNA, referred to as small activating RNA (saRNA), are involved in the recently described phenomenon called RNA activation (RNAa). Recent findings show that saRNA can inhibit cell proliferation and viability via up-regulation of p21(WAF1/CIP1) (p21) in human bladder cancer cells. In the present study, we demonstrate that induction of E-cadherin expression by saRNA leads to suppression of migration and invasion of 5637 human bladder cancer cells in vitro. The elevated E-cadherin expression was confirmed at transcriptional and protein levels after transfection of a 21-nucleotide dsRNA targeting the E-cadherin promoter (dsEcad). Furthermore, this inhibitory effect was associated with relocalization of beta-catenin from the nucleus to the plasma membrane and decreased beta-catenin-mediated transactivation. These data suggest that activation of E-cadherin by saRNA may have a therapeutic benefit for bladder and other types of cancer.